Background: The pathogenesis of rheumatoid arthritis (RA) is predominantly attributed to an autoimmune reaction against citrullinated proteins, coupled with aberrations in T-cell differentiation, which are influenced by a confluence of genetic predispositions and environmental factors.

Objectives: To identify increased immune cell subsets in RA patients via comprehensive immune phenotyping, covering T cells, B cells, NK cells, dendritic cells, and monocyte subsets, and to investigate the implications of these cellular anomalies.

Methods: We conducted immunophenotyping of RA patients’ blood using multi-color flow cytometry and investigated factors contributing to the abnormal cell differentiation in vitro.

Results: In a comparative study involving 533 patients diagnosed with RA who had never used b/tsDMARDs and 96 healthy individuals, we observed alterations primarily centered around abnormal T-cell differentiation in the patients with RA, with a marked increase in CD4 ⁺ effector memory T cells re-expressing CD45RA (CD4 ⁺ TEMRA) cells. To explore the role of these cells in pathogenesis, CD4 ⁺ TEMRA (CD4 ⁺ CCR7 ^- CD45RA ⁺ ) cells were isolated from peripheral blood for transcriptome analysis. These analyses revealed that CD4 ⁺ TEMRA cells exhibited a high expression of cytotoxic molecules and displayed cytotoxic CD4 ⁺ cytotoxic T lymphocytes (CD4 ⁺ CTL)-like functions (Figure 1). Additionally, they prominently expressed p16, a marker indicative of cellular senescence. Furthermore, analysis of the T-cell receptor (TCR) repertoire of these cells revealed a reduced diversity in the TCRs of CD4 ⁺ TEMRA cells compared to both CD4 ⁺ naïve T cells and CD4 ⁺ memory T cells, across both the α and β chains. This suggests an oligoclonal expansion of CD4 ⁺ TEMRA cells. It has been demonstrated that the inclusion of hydrophobic amino acids (such as tryptophan, phenylalanine, and leucine) in the CDR3 region of TCRs in T cells may facilitate the recognition of self-antigens. Consequently, we analyzed the amino acid sequences constituting the TCRs and discovered a frequent utilization of hydrophobic amino acids in the TCRs of CD4 ⁺ TEMRA cells. TCR of regulatory T cells (Tregs) are known for self-reactivity, and when compared to the TCRs of Tregs, CD4 ⁺ TEMRA cells demonstrated a high degree of homology with these TCRs. However, subsequent analysis using Foxp3 and CD25 staining to detect the percentage of Tregs revealed that CD4 ⁺ TEMRA cells contained almost no Tregs. In other words, it was suggested that the CD4 ⁺ TEMRA population exhibits oligoclonal expansion and autoreactivity. In vitro experiments, when naïve T cells were stimulated through TCR activation, some were induced to differentiate into CD4 ⁺ TEMRA cells, expressing both cell senescence markers (p16 and CD57) and cytotoxic molecules. When the telomere length of these CD57-positive cells was detected by PCR, a shortening was observed, confirming that CD4 ⁺ TEMRA were senescent cells. When IL-6 was added to TCR stimulation, the differentiation into CD4+ TEMRA was notably accelerated, increasing from 3.7% to 15.1% (Figure 2). This enhancement was reversed by the administration of tocilizumab and JAK inhibitors. Conversely, IFNγ, IL-12, and IL-17 had no impact on this differentiation. Finally, we investigated the expression of p16 in patients with RA following the administration of molecular targeted therapies. Notably, the expression of p16 in T cells was reduced by the IL-6 inhibitor.

Conclusion: Our study identified an elevation of CD4 ⁺ TEMRA cells in RA patients and highlighted the pivotal role of IL-6 in inducing cellular senescence in T cells and promoting their differentiation into autoreactive phenotypes. These findings emphasize the potential therapeutic benefits of targeting IL-6 to mitigate its effects on T cell aging and differentiation, thereby addressing a critical pathway in RA pathogenesis.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Satoshi Kubo has received speaking fees from Eli Lilly, Bristol-Myers, GlaxoSmithKline, Abbvie, and also research grants from Daiichi-Sankyo, Abbvie, Boehringer Ingelheim, and Astellas, Katsuhide Kusaka: None declared, YUYA FUJITA: None declared, Nishino Takahiro: None declared, Yusuke Miyazaki has received consulting fees from Astra-Zeneca, speaking fees, and/or honoraria from Eli Lilly and GlaxoSmithKline, Kazuyoshi Ishigaki: None declared, Michihiro Kono: None declared, Hiroaki Tanaka: None declared, Hidenori Sakai: None declared, Masanobu Ueno: None declared, Yasuyuki Todoroki: None declared, Yurie Satoh-Kanda: None declared, Ippei Miyagawa: None declared, Shingo Nakayamada has received speaking fees from Bristol-Myers, UCB, Astellas, Abbvie, Eisai, Pfizer, and Takeda, and also research grants from Mitsubishi-Tanabe, Novartis, and MSD, Yoshiya Tanaka has received consulting fees, speaking fees, and/or honoraria from Abbvie, Daiichi-Sankyo, Chugai, Takeda, Mitsubishi-Tanabe, Bristol-Myers, Astellas, Eisai, Janssen, Pfizer, Asahi-Kasei, Eli Lilly, GlaxoSmithKline, UCB, Teijin, MSD, and Santen, and also research grants from Mitsubishi-Tanabe, Takeda, Chugai, Astellas, Eisai, Taisho-Toyama, Kyowa-Kirin, Abbvie, and Bristol-Myers. Hiroaki Tanaka, Yuya Fujita, Hidenori Sakai, Masanobu Ueno, Yasuyuki Todoroki, Yurie Satoh-Kanda, and Ippei Miyagawa have nothing to declare.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.